These studies investigate the role of tyrosine kinases in mediating the differentiation and function of NKT cells, an unusual lymphoid lineage. NKT cells are a unique population with characteristics of both natural killer (NK) cells and T lymphocytes. They represent an important immunoregulatory innate immune population. NKT cells are capable of suppressing a number of deviant immune responses, including graft versus host disease, autoimmunity, and tumor metastasis. Most NKT cells are selected by CD1d rather than classic MHC molecules and utilize an identical TCR alpha chain. Our previous work established that ontogeny of NKT cells differed substantially from NK and T cells. We demonstrated that mutant mice lacking the Src family tyrosine kinase, Fyn, have highly reduced NKT cell numbers, while other lineages were unaffected. Thus our central hypothesis is that Src kinases integrate signals to regulate proper differentiation and function of NKT cells. We will continue investigating the role of Src kinases in regulating development and function of NKT cells. Preliminary data suggests that unlike conventional T lymphocytes, NKT cell function does not depend on Fyn and Lck. Moreover, other Src kinases may be present in NKT cells. The role of other family members in NKT cell function will be explored using mice that are deficient in a variety of Src kinases. The second major area of focus will be to identify effectors that work in concert with Src kinases to regulate NKT cell ontogeny and function. Specifically the role of the signaling adapter molecule, SAP, in directing NKT cell development will be examined. We had demonstrated that SAP plays a critical role early in NKT cell ontogeny, and now will explore the mechanism by which it regulates ontogeny as well as NKT cell function. In addition, the role of Tec family tyrosine kinases in promoting maturation of NKT cells will be examined. Itk deficient NKT cells exhibit a delay in maturation, suggesting that other Tec kinases such as Rlk may facilitate development. The rlk/itk double mutants exhibit defects in both positive arid negative selection of conventional T cells. It is hypothesized that mice deficient in both Rlk and Itk will exhibit highly compromised NKT cell development and defects in negative selection. Together these studies should provide substantial new insight into the pathways regulating NKT cell development. Because of their immunoregulatory potential, a clearer understanding of the mechanisms underlying NKT cell ontogeny and regulation will be of paramount importance for manipulating NKT cells therapeutically. Such approaches could be geared to enhance (cancer immunotherapy) or suppress (airway hypersensitivity) NKT cell function. [unreadable] [unreadable] [unreadable] [unreadable]